HRE和hTERT修饰条件复制型腺病毒携带Egr-1介导的Smac对人食管癌细胞Eca109周期和凋亡的影响

Effects on cell cycle and apoptosis of HRE and hTERT-modified replicative adenovirus carrying Smac mediated by Egr-1 in human esophageal cancer Eca109 cells

目的探讨以HRE和hTERT修饰条件复制型腺病毒携带Egr-1介导的Smac(CARd.pE-Smac)对人食管癌细胞Eca109周期和凋亡的影响。方法利用氯化钴进行化学乏氧,病毒感染滴度为5MOI[Multiplicity of infection(virus/cell)]感染人食管癌Eca109细胞24h,并进行2Gy照射,12h后分别利用Western blotting检测Smac蛋白的表达,PI染色和AnnexinⅤ-FITC双染,流式细胞术检测细胞周期和细胞凋亡变化。实验分为control、CARd.pESmac、hypoxia、2Gy、H+CARd.pE-Smac、CARd.pE-Smac+2Gy和H+CARd.pE-Smac+2Gy组。结果 CARd.pE-Smac感染常氧和乏氧的Eca109细胞后均未见Smac蛋白表达增加,而2Gy照射后,常氧、感染CARd.pE-Smac和乏氧再感染CARd.pE-Smac均能使Smac蛋白表达增加,尤其以三者联用后Smac表达增加最大;感染CARd.pESmac未对细胞周期有明显改变,而乏氧、2Gy和感染CARd.pE-Smac任二者(乏氧与2Gy除外)或者三者联用均能显著增加S期和G2/M期细胞百分比增加(P<0.05,P<0.01),尤其以三者联用作用更强;而且,对于细胞凋亡的诱导作用与周期进程变化的规律相类似。结论 HRE和hTERT修饰条件复制型腺病毒携带Egr-1介导的Smac在人食管癌细胞Eca109中显著过表达,且能够导致细胞发生S期延迟和G2/M期阻滞,并诱导细胞发生凋亡。

Objective To explored the effects on cell cycle and apoptosis of HRE and hTERT-modified replicative adenovirus carrying Smac mediated by Egr-1 （CARd.pE-Smac）in human esophageal cancer Eca109 cells.Methods Chemical hypoxia was achieved by CoCl2 ,human esophageal cancer Eca109 cells were infected with CARd.pE-Smac by infection titer of 5 MOI,after 24 h cells were irradiated by 2 Gy,then after 12 h,cells were collected.Smac protein was measured by Western blotting,cell cycle change and apoptosis were measured by flow cytometry with PI staining and Annexin Ⅴ-FITC double staining,respectively.All groups included control,CARd.pE-Smac,hypoxia,2 Gy,H ＋CARd.pE-Smac,CARd.pE-Smac＋2 Gy and H＋CARd.pE-Smac＋2 Gy.Results After hypoxic and normoxic cells were infected with CARd.pE-Smac,there were not obvious Smac expression increasing,but after 2 Gy irradiation,Smac expression increased in cells of normaxia,CARd.pE-Smac infection and hypoxia＋CARd.pE-Smac infection,especially, Smac expression reached to maximum under three combination.Cell cycles had not obvious changes after CARd.pE-Smac infecting cells,but hypoxia,2 Gy and CARd.pE-Smac infection,as the two （except hypoxia and 2 Gy）or any three party,significantly increased S and G2/M cell percentages （P 〈0.05,P 〈0.01）,especially,its role was most strong in combination with the three;in addition,apoptotic induction regularity was similar with cell cycle changes.Conclusion HRE and hTERT-modified replicative adenovirus carrying Smac mediated by Egr-1 caused Smac obvious overexpression in human esophageal cancer Eca109 cells,and leaded S phage delay and G2/M arrest,and also induced apoptosis.